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Receptor alterations in Huntington’s disease
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Neuroscience Vol. 97, No. 3, pp. 505–519, 2000
505
Copyright 䉷 2000 IBRO. Published by Elsevier Science Ltd
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THE PATTERN OF NEURODEGENERATION IN HUNTINGTON’S DISEASE: A
COMPARATIVE STUDY OF CANNABINOID, DOPAMINE, ADENOSINE AND
GABAA RECEPTOR ALTERATIONS IN THE HUMAN BASAL GANGLIA IN
HUNTINGTON’S DISEASE
M. GLASS,*† M. DRAGUNOW*† and R. L. M. FAULL*‡
Departments of *Anatomy with Radiology, and †Pharmacology, University of Auckland, Private Bag 92019, Auckland, New Zealand
Abstract—In order to investigate the sequence and pattern of neurodegeneration in Huntington’s disease, the distribution and
density of cannabinoid CB1, dopamine D1 and D2, adenosine A2a and GABAA receptor changes were studied in the basal ganglia in
early (grade 0), intermediate (grades 1, 2) and advanced (grade 3) neuropathological grades of Huntington’s disease. The results
showed a sequential pattern of receptor changes in the basal ganglia with increasing neuropathological grades of Huntington’s
disease. First, the very early stages of the disease (grade 0) were characterized by a major loss of cannabinoid CB1, dopamine D2 and
adenosine A2a receptor binding in the caudate nucleus, putamen and globus pallidus externus and an increase in GABAA receptor
binding in the globus pallidus externus. Second, intermediate neuropathological grades (grades 1, 2) showed a further marked
decrease of CB1 receptor binding in the caudate nucleus and putamen; this was associated with a loss of D1 receptors in the caudate
nucleus and putamen and a loss of both CB1 and D1 receptors in the substantia nigra. Finally, advanced grades of Huntington’s
disease showed an almost total loss of CB1 receptors and the further depletion of D1 receptors in the caudate nucleus, putamen and
globus pallidus internus, and an increase in GABAA receptor binding in the globus pallidus internus.
These findings suggest that there is a sequential but overlapping pattern of neurodegeneration of GABAergic striatal efferent
projection neurons in increasing neuropathological grades of Huntington’s disease. First, GABA/enkephalin striatopallidal neurons
projecting to the globus pallidus externus are affected in the very early grades of the disease. Second, GABA/substance P
striatonigral neurons projecting to the substantia nigra are involved at intermediate neuropathological grades. Finally, GABA/
substance P striatopallidal neurons projecting to the globus pallidus internus are affected in the late grades of the disease. In
addition, the finding that cannabinoid receptors are dramatically reduced in all regions of the basal ganglia in advance of other
receptor changes in Huntington’s disease suggests a possible role for cannabinoids in the progression of neurodegeneration in
Huntington’s disease. 䉷 2000 IBRO. Published by Elsevier Science Ltd.
Key words: receptor changes, caudate nucleus, putamen, globus pallidus, substantia nigra.
GPi. 1,16,49 By late grades of Huntington’s disease, all striatal
projection neurons show extensive loss. In the present study
the validity of this proposed pattern of neuronal degeneration
in Huntington’s disease has been investigated by studying
changes in the binding of a range of neurotransmitter receptors, including the CB1 cannabinoid receptor, 39 in the basal
ganglia of Huntington’s disease patients.
Receptor binding studies in the human and rat brains have
demonstrated that cannabinoid receptors are presynaptically
localized on striatonigral and striatopallidal terminals in the
SN and globus pallidus. 25,30,37 These findings, together with
the demonstration that D1 receptors in the SN and GPi
regions, and, D2 and A2a receptors in the GPe region 21,34,59
are presynaptically localized on striatal efferent terminals
suggest the possibility that cannabinoid receptors are colocalized with these various types of receptors in the SN
and globus pallidus. Also, the well defined co-localization
of the cannabinoid CB1, dopamine D1, dopamine D2 and
adenosine A2a receptors in the caudate nucleus and putamen
has enabled us to compare and contrast the receptor changes
in the early and late grades of Huntington’s disease in order to
provide further information on the sequence and pattern of
neurodegeneration in Huntington’s disease.
Huntington’s disease is characterized by an atrophy of the
caudate nucleus and putamen. 27 Medium spiny GABAergic
striatal projection neurons, the predominant neostriatal cell
type, are particularly vulnerable in Huntington’s disease, 27
while there is selective sparing of cholinergic interneurons, 18,32
and interneurons containing somatostatin, neuropeptide Y,
and NADPH-diaphorase. 10,19
Two populations of GABAergic striatal efferent neurons
can be demonstrated based on their projection targets and
neuropeptide content. 6,22,46,50 Striatal neurons projecting to
the globus pallidus externus (GPe) are enriched in metenkephalin (enk), whereas the striatal neurons projecting to
the globus pallidus internus (GPi) and to the substantia nigra
(SN) are enriched in substance P. 50 Recent studies have
suggested a differential pattern of degeneration of these
projection neurons in Huntington’s disease, with GABA/
enk-containing neurons projecting to the GPe and GABA/
substance P-containing striatal neurons projecting to the SN
being preferentially affected in pre-symptomatic cases and in
early degenerative grades of Huntington’s disease, with relative
sparing of GABA/substance P-containing neurons projecting to
‡To whom correspondence should be addressed. Tel.: ⫹ 64-9-3737599
(ext. 6708); fax: ⫹ 64-9-3737484.
E-mail address: [email protected] (R. L. M. Faull).
Abbreviations: enk, enkephalin; FNZ, flunitrazepam; GPe, globus pallidus
externus; GPi, globus pallidus internus; NADPH, reduced nicotinamide
adenine dinucleotide phosphate; SN, substantia nigra.
EXPERIMENTAL PROCEDURES
Tissue collection
The human brain tissue used in these studies was obtained from the
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M. Glass et al.
New Zealand Neurological Foundation Human Brain Bank in the
Department of Anatomy, University of Auckland and the study was
approved by the University of Auckland Human Subjects Ethics
Committee.
All control subjects had previously been in good health with no
known history of neurological disease or drug treatment and all had
died suddenly without the opportunity of receiving any form of medical treatment. For both control and Huntington’s disease cases, the
brains were removed to the Department of Anatomy, University of
Auckland, immediately following autopsy. On arrival, tissue blocks
were immediately selected from various regions of the basal ganglia.
The tissue blocks were frozen on dry ice and stored at ⫺80⬚C prior to
subsequent autoradiographical processing as detailed below. The post
mortem delay in each case is described as the time interval between
death and the freezing of the tissue blocks.
The control tissue consisted of post mortem human brains obtained
from six adult subjects (aged 21–81 years; average age 59 years;
average post mortem delay 10 h; see Table 1 for details). The
Huntington’s disease tissue was obtained from 10 patients diagnosed
with Huntington’s disease, and graded according to the five point (0–4)
neuropathological grading scale criteria of Vonsattel and colleagues 43,67
(two subjects were grade 0, three subjects grade 1, three subjects grade
2, and two subjects grade 3; see Table 2 for details). The subjects
ranged in age from 56–87 years, average age 63 years; average post
mortem delay 16 h.
Autoradiography
For these studies frozen blocks of unfixed tissue were mounted on to
cryostat chucks and 16-mm sections were thaw mounted on to gelatine/
chrome-alum-coated slides. Sections were stored at ⫺80⬚C until
labelled.
All autoradiographical techniques have been previously
described. 11,14,25 For each ligand used, triplicate sections from relevant
regions of each brain were labelled. In brief, cannabinoid CB1 receptors were labelled at 2.5 nM with [ 3H]CP55,940 (Dupont/NEN; specific activity, 125 Ci/mmol). The sections were incubated for 2 h at 37⬚C
in 50 mM Tris–HCl buffer (pH 7.4) with 5% bovine serum albumin.
The sections were then washed twice at 4⬚C in 50 mM Tris buffer with
1% bovine serum albumin for 2 h. Non-specific binding was determined by incubation in the presence of 10 mm CP55,940. Dopamine
D1 receptors were identified using 1 nM [ 3H]SCH23390 (Dupont/
NEN; specific activity, 80.4 Ci/mmol) in 50 mM Tris–HCl buffer
(with 1 mM MgCl2, 2 mM CaCl2, 5 mM KCl, and 120 mM NaCl,
pH 7.4); the sections were incubated for 30 min at room temperature
before being rinsed twice for 5 min in ice-cold buffer. Non-specific
binding was determined by incubation in the presence of 1 mM dopamine. Dopamine D2 receptors were labelled for 20 min at room
temperature in 3 nM [ 3H]Raclopride (Dupont/NEN; specific activity,
79.5 Ci/mmol) in 170 mM Tris–HCl buffer (with 1 mM MgCl2, 2 mM
CaCl2, 5 mM KCl, and 120 mM NaCl, pH 7.7); the sections were
rinsed four times for 1 min each in ice-cold buffer. Non-specific binding was determined by incubation in the presence of 1 mM dopamine.
Sections for adenosine A2a binding were preincubated for 30 min in
1 U/ml adenosine deaminase (Sigma, type IV) in 50 mM Tris–HCl
buffer (with 10 mM MgCl2, pH 7.4), before labelling with 5 nM
[ 3H]CGS21680 (Dupont/NEN; specific activity, 42.6 Ci/mmol) for
2 h; the sections were then rinsed and washed twice for 5 min in buffer
before being rinsed in ice-cold distilled H20. Non-specific binding was
determined by incubation in the presence of 20 mM 2-chloroadenosine.
GABAA receptors were labelled using 1 nM [ 3H]flunitrazepam (FNZ,
Amersham; specific activity, 84 Ci/mmol) in 50 mM Tris–HCl buffer,
pH 7.4; the sections were incubated for 1 h at 4⬚C and then washed
twice for 1 min in ice-cold buffer before being rinsed in ice-cold
distilled H2O. Non-specific binding was determined by incubation in
the presence of 1 mM FNZ. All sections were fan-dried at 4⬚C overnight and placed in X-ray cassettes with tritium-microscale calibration
slides (Amersham), where they were exposed to tritium-sensitive
hyperfilm for 10 weeks prior to developing. Integrative density
measurements of each region were made using the MD30 Image
Analysis System (Leading Edge Pty, Australia). The binding in the
Huntington’s disease brains is presented as a percentage of the mean
of the binding measured in control brains. For Grade 1 and 3 the data
are presented as the mean percentage difference ^ S.E.M. For Grade 0
and 3, where there were only two cases, the mean percentage difference for each case were averaged and are presented with their errors.
Table 1. Source of control post mortem human brain tissue
Case
Sex
Age
(years)
H47
H78
H79
H80
H81
H82
M
F
M
M
M
M
81
48
75
72
55
21
Post mortem
delay (h)
6.5
11.5
11
10
12
8.5
Cause of death
Subarachnoid haemorrhage
Coronary artery disease
Myocardial infarction
Myocardial infarction
Myocardial infarction
Carbon monoxide poisoning
Table 2. Source of post mortem Huntington’s disease brain tissue
Case
Sex
Age
(years)
Post mortem
delay (h)
HD grade
(CAG)n in IT15
HC46
M
59
2.5
0
16/43
HC66
HC55
HC51
HC53
HC52
HC57
HC61
HC58
HC48
M
M
M
M
F
M
M
M
M
62
87
58
56
61
58
65
64
62
19
20
4.5
14
23
29
6
19
20
0
1
1
1
2
2
2
3
3
27/41
14/42
16/43
17/43
19/46
18/46
18/47
18/44
17/47
Cause of death
Chronic obstructive
respiratory disease
Pneumonia
Perforated duodenal ulcer
Pneumonia
Bowel obstruction
Myocardial infarction
Myocardial infarction
Pneumonia
Pneumonia
Septicemia
Abbreviations used in the figures and tables
CN
ENK
HD
PU
caudate nucleus
enkephalin
Huntington’s disease
putamen
SNc
SNr
SP
VS
substantia nigra pars compacta
substantia nigra pars reticulata
substance P
ventral striatum
Receptor alterations in Huntington’s disease
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Fig. 1. Autoradiograms showing the binding of [ 3H]CP55,940 to cannabinoid CB1 receptors in the caudate nucleus and putamen of: (A) control; (B) grade 0
Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. There is a moderate decrease in CB1 receptor binding at
grade 0 (B) with a further marked loss of receptors at more advanced grades of Huntington’s disease (C, D). Scale bar ˆ 1 cm.
RESULTS
The principal aim of this study was to investigate the
pattern of cannabinoid CB1, dopamine D1 and D2, adenosine
A2a and GABAA receptor changes in the basal ganglia in the
human brain in early (grade 0), intermediate (grade 1, 2) and
late (grade 3) neuropathological grades of Huntington’s
disease in order to gain further information on the possible
neuronal co-localization of these receptors in the human basal
ganglia and on the sequence and pattern of neurodegeneration in Huntington’s disease. The various receptors were
demonstrated in the basal ganglia using receptor autoradiography following in vitro labelling of cryostat sections with
tritiated ligands specific for the various receptor subtypes.
As shown in Figs 1–10, the pattern and density of autoradiographic receptor labelling for each of the receptors in the
various nuclei of the basal ganglia—caudate nucleus, putamen, GPe, GPi and SN—were compared between control
brains and early, intermediate and late stage Huntington’s
diseased brains. The density of the receptors in each of the
nuclei in the basal ganglia was then determined using computerized densitometry methods (Tables 3–7). For all of the
receptors studied the values observed in the control brains
were comparable to previously reported values. 7,9,15,24,26,38,66
The results on the various types of receptors studied are
detailed below.
Cannabinoid CB1 receptors
The caudate nucleus and putamen, showed a moderately
low level of cannabinoid CB1 receptor binding in the normal
brain (Figs 1A, 2A). As described previously, 24 careful examination of the pattern of receptor labelling in the caudate
nucleus and putamen suggests a patchy distribution of receptors, especially in the caudal putamen at the level of the
lenticular nucleus (Fig. 2A). The grade 0 Huntington’s
disease cases (Figs 1B, 2B) exhibited a moderate decrease
in cannabinoid receptor binding (46–52%; Table 3) as
compared to controls (Figs 1A, 2A). The cannabinoid receptor binding decreased dramatically in all Huntington’s disease
cases with more advanced pathology, that is, grade 1 and
greater (Table 3). The grade 1 cases exhibited an average
level of binding of only 21–31% of the normal (Figs 1C,
2C; Table 3), and further decreases were observed within
the grade 2 and 3 cases, which exhibited binding similar to
background levels.
Very high densities of cannabinoid receptor binding sites
were seen in the globus pallidus of the control brains (Fig.
2A). The highest densities of receptors were present in the
GPi and moderate densities of receptors were present
throughout the rostrocaudal extent of the globus pallidus
externus (Fig. 2A). Closer examination of the pattern of
autoradiographic receptor labelling in the GPe revealed
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M. Glass et al.
Fig. 2. Autoradiograms showing the binding of [ 3H]CP55,940 to cannabinoid CB1 receptors in the putamen and globus pallidus of the lenticular nucleus of: (A)
control; (B) grade 0 Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. In the putamen there is an increasing
loss of CB1 receptor binding from grade 0 (B) to advanced grades (C, D) with an almost total loss of receptors at grade 3 (D). In the globus pallidus there is a
differential loss of receptors with increasing neuropathological grades of Huntington’s disease; CB1 receptor binding is almost totally lost at grade 0 in the GPe
(B), but is not totally lost in the GPi until grade 3 (D). Scale bar ˆ 1 cm.
some regional variations in the density and pattern of receptor
binding; higher density patches appeared to be present in
some regions, with the highest density of labelling being
present in the rostrolateral region of the complex and with
lower densities of binding in the ventral pallidum.
Cannabinoid receptor binding was decreased dramatically
in both pallidal segments in all cases of Huntington’s disease
(Fig. 2B–D). Within the very early stages of Huntington’s
disease (grade 0, Fig. 2B), the loss of CP55,940 binding
was pronounced in the globus pallidus externus and density
measurements showed that binding densities in GPe were
reduced to 9% of normal (Table 3). In contrast, as shown in
Table 3, the density of CB1 binding in GPi had reduced to
Table 3. Cannabinoid CB1 receptor levels in Huntington’s disease brains—
results are given as a percentage of the binding in control cases
[ 3H]CP55,940—% of control levels
HD grade
0
1
2
3
CN
PU
GPe
GPi
SNr
46 ^ 14
21 ^ 4
9^5
8^3
52 ^ 17
31 ^ 5
20 ^ 6
8^2
9^2
15 ^ 1
3^2
7^3
19 ^ 4
14 ^ 3
3^1
4^2
19 ^ 3
10 ^ 7
9^2
4^2
19% of normal. However, in the more advanced cases of
Huntington’s disease (Fig. 2C–D), receptor binding in both
segments had dramatically decreased to an average of
between 3–7% of normal levels (Table 3).
As described previously, 24,25 cannabinoid receptor labelling
within the SN was very dense and discreetly localized to the
pars reticulata. As shown in Fig. 7A–C and Table 3, the levels
of cannabinoid binding showed a marked decrease in grade 0
(19% of normal), and even greater decreases by grade 1 (10%
of normal). By grade 2, binding was undetectable above background levels.
Dopamine D1 and D2 receptors
Within the caudate nucleus and putamen a fairly homogeneous distribution of dopamine D1 (Figs 3A, 4A) and D2
(Figs 5A, 6A) receptors was observed in the control brains. At
grade 0 Huntington’s disease, normal levels of D1 receptor
binding were present in the caudate nucleus and putamen
(Figs 3B, 4B; Table 4), while a major loss of D2 receptor
binding was observed (Figs 5B, 6B; Table 5, average of
40–44% of normal). In grade 1 cases, the density of D2 receptors in the caudate nucleus and putamen had further reduced
to 6–7% of normal (Figs 5C, 6C; Table 5) and D1 receptors
showed a moderate decrease to 54–56% of normal (Table 4,
Receptor alterations in Huntington’s disease
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Fig. 3. Autoradiograms showing the binding of [ 3H]SCH23390 to dopamine D1 receptors in the caudate nucleus and putamen of: (A) control; (B) grade 0
Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. Grade 0 (B) showed generally normal levels of D1
receptor binding but there was some evidence of a “patchy” loss of receptors in regions of the caudate nucleus and putamen. There was an increasing loss of D1
receptor binding at more advanced grades of Huntington’s disease with a further marked “patchy” loss of receptors (C, D). Scale bar ˆ 1 cm.
Table 4. Dopamine D1 receptor levels in Huntington’s disease brains—
results are given as a percentage of the binding in control cases
[ 3H]SCH23390—% of control levels
HD grade
0
1
2
3
Table 5. Dopamine D2 receptor levels in Huntington’s disease brains—
results are given as a percentage of the binding in control cases
[ 3H]Raclopride—% of control levels
HD grade
CN
PU
GPe
GPi
SNr
115 ^ 19
54 ^ 20
34 ^ 7
26 ^ 9
118 ^ 18
56 ^ 14
28 ^ 14
32 ^ 11
–
–
–
–
106 ^ 7
100 ^ 3
34 ^ 20
6^2
74 ^ 6
80 ^ 18
31 ^ 8
11 ^ 5
Figs 3C, 4C). In more advanced Huntington’s cases (grades 2
and 3), the density of D2 receptors in the caudate nucleus and
putamen was barely above background levels (6–10% of
normal; Figs 5D, 6D), and D1 receptor densities further
reduced to 26–34% of normal (Table 4; Figs 3D, 4D). Of
particular interest was the finding that the loss of dopamine
receptor binding within the caudate nucleus and putamen was
not homogeneous. Irregularly shaped patches of both the
caudate nucleus and putamen exhibited greater D1 and D2
binding loss than adjacent areas, giving the autoradiograms
a “patchy” appearance (see Figs 3 and 5). This “patchy”
pattern of receptor loss appeared reminiscent of the striosome/matrix compartmentation previously described for
various neurochemical markers in the human caudate nucleus
and putamen (see Ref. 28 for review). Furthermore, even
0
1
2
3
CN
PU
GPe
GPi
44 ^ 22
6^2
21 ^ 7
10 ^ 1
40 ^ 21
7^3
12 ^ 4
10 ^ 2
6^4
4^4
1^1
–
–
–
–
–
in grade 3 Huntington’s disease, a sub-population of D1
receptors was preserved (Fig. 3D), while little D2 binding
was visible (Fig. 5D).
Within the normal globus pallidus, moderately low levels
of D1 receptors were located in GPi only (Fig. 4A), while
moderate levels of D2 receptors were present in the GPe
(Fig. 6A). All Huntington’s disease grades show a dramatic
loss of D2 receptor binding in GPe. In particular, dopamine D2
receptors in the GPe show a dramatic reduction in the very
early stages of Huntington’s disease; in grade 0 brains D2
receptor binding in GPe is reduced to 40–44% of controls,
and, in grade 1 (reduced to 6–7% of control) and more
advanced cases, D2 labelling is barely above background
levels (Fig. 6; Table 5). In contrast, the density of D1 receptor
binding in the GPi of grade 0 and grade 1 Huntington’s
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M. Glass et al.
Fig. 4. Autoradiograms showing the binding of [ 3H]SCH23390 to dopamine D1 receptors in the putamen and globus pallidus of the lenticular nucleus of: (A)
control; (B) grade 0 Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. In the putamen, there is an increasing
“patchy” loss of D1 receptor binding from grade 0 (B) to advanced grades (C, D). As in the control, there is an absence of D1 receptors in the GPe at all
neuropathological grades. In the GPi, D1 receptor binding density at grades 0 (B) and 1 (C) is similar to the control, but is markedly reduced at grade 3 (D).
Scale bar ˆ 1 cm.
disease brains was equivalent to binding in the control
pallidum (Table 4); intermediate levels of D1 receptor binding
were present at grade 2, while in the grade 3 cases, D1 receptor
binding was barely detectable (Fig. 4D; Table 4).
Within the SN only D1 receptors were examined, as only
very low levels of D2 receptors were identified in the SN in the
control brains. In normal control brains, D1 receptors were
discreetly localized within the pars reticulata of the SN
(Fig. 7D). Only a slight loss of D1 receptor binding was
observed in grade 0 and grade 1 Huntington’s disease (74–
80% of control, Table 4; Fig. 7E, F). In the later grades of
Huntington’s disease the loss of receptor binding became
more pronounced with D1 receptor binding barely detectable
above background levels in grade 3 Huntington’s disease
(Table 4).
Adenosine A2a receptors
A2a receptor binding was fairly homogeneous within the
caudate nucleus and putamen of control brains (Figs 8A,
9A). Within the caudate nucleus and putamen a dramatic
loss of adenosine A2a receptor binding was observed in
grade 0 Huntington’s disease cases (34–35% of controls),
and there was a further dramatic decrease in A2a receptor
binding in grade 1 Huntington’s disease to 11–13% of
controls (Figs 8C, 9C; Table 6); more advanced cases showed
no detectable A2a receptor binding (Figs 8D, 9D; Table 6). As
for the dopamine receptors, the binding appeared to decline in
a heterogeneous fashion, with irregularly shaped patches of
receptors declining slightly more rapidly than the receptors in
the surrounding regions (Fig. 8B, C).
In the globus pallidus, adenosine A2a receptors were present
only within the GPe (Fig. 9A). There was a dramatic and total
loss of A2a receptors from GPe in the very earliest stages of
Huntington’s disease; in all grade 0 cases and in all cases of
more advanced pathology there was no detectable adenosine
A2a receptor binding (Fig. 9; Table 6).
GABAA receptors
GABAA receptor binding showed an increasing patchy loss
in the caudate nucleus and putamen in grade 0 (Fig. 10B) and
grade 1 (Fig. 10C) with an almost total loss of receptors in the
caudate nucleus and putamen at more advanced grades of
Huntington’s disease (Fig. 10D). In contrast, GABAA receptor
binding within the globus pallidus showed increased binding
densities with increasing neuropathological grades of
Huntington’s disease (Fig. 10; Table 7). In confirmation of
previous studies 16 a marked up-regulation of [ 3H]FNZ binding was observed within the GPe in grade 0 (156% of control)
Receptor alterations in Huntington’s disease
511
Fig. 5. Autoradiograms showing the binding of [ 3H]Raclopride to dopamine D2 receptors in the caudate nucleus and putamen of: (A) control; (B) grade 0
Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. There was a marked “patchy” decrease in D2 receptor
binding at grade 0 (B) with a further increasing loss of receptors at more advanced grades of Huntington’s disease (C, D). Scale bar ˆ 1 cm.
Table 6. Adenosine A2a receptor levels in Huntington’s disease brains—
results are given as a percentage of the binding in control cases
HD grade
0
1
2
3
CGS21680 binding—% of control levels
CN
PU
GPe
34 ^ 10
13 ^ 7
2^0
6^1
35 ^ 1
11 ^ 6
0
1^1
2^2
0
1^1
0
and this was sustained in Huntington’s disease cases with
more advanced pathology (Fig. 10B–D; Table 7). Up-regulation
of GABAA receptors within the GPi was not observed until
grade 1; this up-regulation was sustained in grade 2 cases
(129%) and further increased (156% of control) in more
advanced grade 3 cases (Table 7).
DISCUSSION
It is now well established that medium spiny neurons of the
caudate nucleus and putamen are preferentially vulnerable in
Huntington’s disease. 27 Furthermore, the subset of the
medium spiny projection neurons containing GABA/enk
demonstrate preferential dysfunction in terminal areas in the
GPe. 49,52,61 In contrast, medium spiny neurons containing
Table 7. GABAA receptor levels in Huntington’s disease brains—results
are given as a percentage of the binding in control cases
HD grade
0
1
2
3
[ 3H]FNZ binding—% of control levels
GPe
GPi
156 ^ 7
129 ^ 2
126 ^ 23
139 ^ 13
106 ^ 4
125 ^ 5
129 ^ 7
156 ^ 10
GABA/substance P projecting to the GPi are more resistant
to dysfunction in early Huntington’s disease. However,
conflicting information on the relative loss of enkephalincontaining terminals versus substance P-containing terminals
exists. 16,58,63 Since cannabinoid, dopamine (D1 and D2) and
adenosine receptors are localized in various combinations
on the cell bodies and terminal axons of striatal efferent
neurons projecting to the GPe, GPi and SN (see Fig.
11A), 21,25,30,34,37,59 the present study has utilized the technique
of receptor autoradiography to examine changes in cannabinoid, dopamine and adenosine receptors in the basal ganglia
in Huntington’s disease brains ranging from pathological
grade 0 to grade 3 in order to further investigate the pattern
of degeneration of striatal efferent neurons in this disease.
All receptors studied demonstrated a greater loss of binding
512
M. Glass et al.
Fig. 6. Autoradiograms showing the binding of [ 3H]raclopride to dopamine D2 receptors in the putamen and globus pallidus of the lenticular nucleus of: (A)
control; (B) grade 0 Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. In the putamen there is a very marked
decrease in D2 receptor binding at grade 0 (B) with a total loss of receptor binding at more advanced neuropathological grades of Huntington’s disease (C, D).
Compared with the control (A), there is a total loss of D2 receptor binding in the GPe at grade 0 (B) and at more advanced grades (C, D), while the GPi shows no
D2 receptor binding in the control (A) and Huntington’s disease (B–D) brains. Scale bar ˆ 1 cm.
within the projection regions than within the caudate nucleus
and putamen itself in early grade Huntington’s disease, a
finding consistent with a previous study by Richfield and
Herkenham. 53 As suggested by these authors, two possible
processes can explain this observation. First, it may represent
perikaryal dysfunction associated with deficient production,
processing or transport of receptors to terminals. Secondly,
loss of receptors in the pallidum may reflect primary dysfunction in terminals followed by retrograde degeneration of
projection neurons. Interestingly, presymptomatic cases
demonstrate loss of enkephalin immunoreactivity in GPe,
but preservation of enkephalin-containing neurons in the
caudate nucleus and putamen, supporting primary terminal
dysfunction. 52
The results of this study indicate that the medium spiny
neurons exhibit a selective vulnerability in early Huntington’s
disease. Figure 11 demonstrates the overall pattern of degeneration of the neurons, their terminals and the receptors within
the basal ganglia as suggested by this study. The results show
that, in agreement with previous studies, the medium spiny
neurons in the caudate nucleus and putamen comprise of at
least three different populations of GABAergic neurons: those
containing enkephalin projecting to GPe; and two populations
containing substance P, one projecting to the GPi and the
other to the SN. While there may be some overlap within
these populations, each group appears to have a different
vulnerability to the disease process. Selective vulnerability
was particularly indicated by the differential loss of dopamine
D1 and D2 receptor binding. Binding to both of these receptors
declined in a heterogeneous fashion from sub-populations of
neurons, giving the autoradiograms a “patchy” appearance.
The regions of binding were not discreet for D1 and D2
receptors but rather appeared to overlap in many regions. A
similar finding was observed by Richfield et al. 54 in early
Huntington’s disease cases. What is particularly interesting
to note in this study is the much more rapid loss of dopamine
D2 receptors as opposed to D1 receptors, a finding which is
contrary to earlier results. 54 Since D2 receptors are believed to
be localized predominantly on GABA/enk containing neurons
which project to GPe, while D1 receptors are localized to
GABA/substance P-containing neurons projecting to GPi
and SN pars reticulata, this finding therefore confirms
previous studies of presymptomatic Huntington’s disease
allele carriers, where immunohistochemical results demonstrated that degeneration of striatal neurons projecting to
GPe occurs earlier in the course of the disease than loss of
neurons projecting to GPi, 1,49 a finding which has been further
supported by other studies in early grade Huntington’s
disease. 16,58 Furthermore, the loss of dopamine D1 receptor
binding within the SN at grade 1, when levels within GPi
Receptor alterations in Huntington’s disease
513
Fig. 7. Autoradiograms showing the distribution of cannabinoid CB1 (A–C) and dopamine D1 (D–F) receptors in the SN of control (A, D) and Huntington’s
disease (B, C, E, F) brains. The autoradiograms demonstrate the binding of [ 3H]CP55,940 (A–C) to cannabinoid CB1 receptors and [ 3H]raclopride (D–F) to
dopamine D1 receptors in the SN in control (A, D); grade 0 Huntington’s disease (B, E) and grade 1 Huntington’s disease (C, F) brains. Cannabinoid CB1
receptor binding in the SN shows very high densities in control brains (A); CB1 receptor binding in the SN is reduced in grade 0 Huntington’s disease (B) and is
almost absent at higher neuropathological grades (C). Dopamine D1 receptor binding in the SN shows no obvious change in grade 0 (E) compared with the
control (D), but binding appears reduced in grade 1 (F) Huntington’s disease cases. Scale bar ˆ 1 cm.
were comparable to control levels, suggests that the population
of GABA/substance P neurons projecting to SN pars reticulata, is distinct from the population of neurons projecting to
GPi. Also, the heterogeneous patchy loss of D1 and D2 dopamine receptors (and adenosine A2a and GABAA receptors) in
the caudate nucleus and putamen in the earlier stages of the
disease is in agreement with previous in situ and immunohistochemical studies by us 5,42 and others 29 suggesting that the
projection neurons in the striosome compartment of the
caudate nucleus and putamen may be especially vulnerable
in early Huntington’s disease.
Within the rat caudate nucleus and putamen it has been
suggested that 15–20% of D1 receptors are localized on
non-medium spiny interneurons; 35 thus it may be that the
surviving D1 receptors present in the caudate nucleus and
putamen in advanced diseased cases are localized on this
subset of interneurons which are still present at Grade 3
Huntington’s disease. This is in agreement with the results
of our previous in situ studies on D1 and D2 receptor gene
expression showing the relative survival of a subset of D1
mRNA-positive neurons in the caudate nucleus and putamen
of advanced Huntington’s disease. 4 In contrast, the almost
total loss of D2 receptors (Figs 3, 4) and D2 mRNA-expressing
neurons 4 within the caudate nucleus and putamen in advanced
Huntington’s disease suggests that, unlike rat caudate nucleus
and putamen, 3 a sub-population of D2 receptors may not be
present on the interneurons believed to be preserved in
Huntington’s disease. 18,32 However, in contrast to this study,
514
M. Glass et al.
Fig. 8. Autoradiograms showing the binding of [ 3H]CGS21680 to adenosine A2a receptors in the caudate nucleus and putamen of: (A) control; (B) grade 0
Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. There is a very marked decrease in A2a receptor binding
in the caudate nucleus and putamen at grade 0 (B) with an almost total loss of receptors at more advanced grades of Huntington’s disease (C, D).
Scale bar ˆ 1 cm.
a previous study, 53 demonstrated 30–40% of normal levels of
D2 receptors in grade 3 Huntington’s disease, supporting the
localization of D2 receptors presynaptically on nigrostriatal
terminals and on interneurons; the reasons for these differences are not clear. The almost total loss of D1 receptor binding within the SN in grade 3 Huntington’s disease confirms
previous findings showing that D1 receptors within the SN are
localized exclusively to the terminals of striatal projection
neurons. 66
The results in this study confirm an earlier study by MartinezMir et al. 38 demonstrating A2a receptor loss in the caudate
nucleus and putamen in Huntington’s disease. The loss of
A2a receptors in the caudate nucleus and putamen in the
present study paralleled the loss of D2 receptors. The similarities in the changes in A2a and D2 receptor binding was
expected, as in situ hybridization studies have demonstrated
that rat A2a adenosine receptors are co-expressed in the same
striatal neurons as D2 dopamine receptors, with no A2a receptors co-expressed with either D1 receptors or substance P. 21,59
The loss of A2a receptors from both GPe and the caudate
nucleus and putamen in grade 0 again confirms the loss of
this subset of medium spiny projection neurons early in the
disease process. Studies in post mortem human brain have
previously suggested that the A2a site may be present on
cholinergic interneurons within the caudate nucleus and
putamen 33 and since the cholinergic interneurons are relatively spared in Huntington’s disease, 18,32 then a proportion
of A2a receptors would be expected to be preserved in the
caudate nucleus and putamen of Huntington’s disease brains.
However, a virtually total loss of A2a binding was observed in
the caudate nucleus and putamen in grades 1–3 Huntington’s
disease suggesting that the A2a receptors are either localized solely to the medium spiny neurons, or that A2a receptors
are localized in part on cholinergic interneurons, and that these
neurons are also vulnerable in early Huntington’s disease.
Recent studies have demonstrated that adenosine A2a receptors inhibit the activity of striatal dopamine D2 receptors by
decreasing their affinity for agonists 20 and by regulating their
gene expression in enkephalinergic neurons. 60 A study by
Popoli et al. 48 demonstrated that CGS21680 exhibits a protective effect on dopamine induced hyperactivity in the quinolinic acid-lesioned rat. The authors of this study therefore
suggested that A2a receptor agonists may be beneficial in
the treatment of Huntington’s disease. In support of this
suggestion, studies have shown that the activation of A2a
receptors can enhance the electrically stimulated release of
GABA in the pallidum. 40 However, loss of receptor binding
in this area may limit the effectiveness of A2a specific drugs,
and furthermore, may be a contributing factor to the disease
symptoms.
Receptor alterations in Huntington’s disease
515
Fig. 9. Autoradiograms showing the binding of [ 3H]CGS21680 to adenosine A2a receptors in the putamen and globus pallidus of the lenticular nucleus of: (A)
control; (B) grade 0 Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. In the putamen, there is a very
marked decrease in A2a receptor binding at grade 0 (B) with a total loss of receptors at more advanced grades of Huntington’s disease (C, D). Compared with the
control (A), there is a total loss of A2a receptor binding in the GPe at grade 0 (B) and at more advanced grades (C, D), while the GPi shows no A2a receptor
binding in the control (A) and Huntington’s disease (B–D) brains. Scale bar ˆ 1 cm.
Within the caudate nucleus and putamen the loss of cannabinoid receptors was in between the loss of D1 and D2 receptors in grade 0 Huntington’s disease, suggesting that
cannabinoid receptors are localized on both GABA/enk and
GABA/substance P projection neurons, as has been demonstrated previously. 37 Within the globus pallidus, cannabinoid
receptor binding was dramatically decreased in the GPe in the
very early Huntington’s disease cases, and exceeded the loss
of binding density within the GPi; this finding is consistent
with GABA/enk neurons projecting to the GPe being more
vulnerable in early Huntington’s disease than GABA/
substance P neurons projecting to the GPi. The selective
vulnerability of striatal-GPe projection neurons is further
supported by the finding that the loss of cannabinoid receptor
binding within the GPe in grade 0 Huntington’s disease is
accompanied by a comparable loss of D2 and A2a receptor
binding in the GPe. These findings therefore suggest that
striatopallidal projection terminals in GPe degenerate at early
stages of Huntington’s disease. This pattern of degeneration is
further supported by the observed up-regulation of GABA
receptors in GPe in the grade 0 cases. These receptors are
postsynaptic in the globus pallidus, and their up-regulation
in Huntington’s disease has been interpreted as a denervation
supersensitivity phenomenon reflecting the loss of GABA input
secondary to the degeneration of striatal neurons. 12,16,17,47,51
In contrast to the receptor changes in the GPe, where
cannabinoid and dopamine D2 receptors were lost simultaneously in Huntington’s disease, in the GPi the cannabinoid
receptor changes preceded alterations in D1 receptor binding.
In grade 0 Huntington’s disease there was a substantial loss of
cannabinoid receptor binding in the GPi. However, in these
cases D1 receptor binding was normal and there was no
evidence of up-regulation of GABAA receptors suggesting
the preservation of the GPi synaptic terminals in these
cases. Thus, in the grade 0 cases there appears to be a preferential loss of cannabinoid receptor binding in GPi prior to
terminal degeneration. In grade 1 Huntington’s disease, the
findings are more complicated. A further decrease in cannabinoid receptor binding is observed, while the density of
D1 receptors remained at normal levels, suggesting intact
terminals. The preservation of striatopallidal terminals is
further supported by normal substance P concentrations in
GPi in Grade 1 cases. 1,16,49 However, an up-regulation of
GABAA receptors is detectable in grade 1 Huntington’s
disease, suggesting that alterations in the functioning of
the medium spiny neurons, in the form of decreased
GABA levels, are occurring prior to any detectable terminal
degeneration.
Consistent with the results in the GPi is the finding of
a similar pattern of changes in the SN. Thus, in grade 0
Huntington’s disease cannabinoid receptors in the SN
demonstrated a pronounced decrease in binding density
516
M. Glass et al.
Fig. 10. Autoradiograms showing the binding of [ 3H]FNZ to GABAA receptors in the putamen and globus pallidus of the lenticular nucleus of: (A) control; (B)
grade 0 Huntington’s disease; (C) grade 1 Huntington’s disease; and (D) grade 3 Huntington’s disease brains. There is a gradual increasing “patchy” loss of
GABAA receptor binding in the putamen at grade 0 (B) and grade 1 (C) with an almost total loss of receptors at advanced grades of Huntington’s disease (D). In
the globus pallidus, there is a marked increase in GABAA receptor binding in the GPe at grade 0 and in both the GPe and GPi at more advanced grades of
Huntington’s disease (C, D). Scale bar ˆ 1 cm.
but D1 receptor binding was equivalent to that seen in
controls. If D1 receptors can be considered to be markers
for striatonigral terminals then these findings would again
suggest that the cannabinoid receptor binding is being
compromised prior to the degeneration of the terminals. It
is difficult to explain the possible functional significance
of the loss of CB1 receptors prior to the loss of co-localized
dopamine receptors. In recent years several excellent
studies have investigated the interactions of cannabinoid
and dopamine in the projection nuclei of the basal ganglia 23,55–57 demonstrating a highly complex interaction
between these two systems. It is interesting to speculate
that perhaps the early down-regulation of cannabinoid receptors is a compensatory mechanism in Huntington’s disease.
Albin et al. 2 proposed a model for the early symptoms of
Huntington’s disease which demonstrates that decreased
GABA/enk input to the GPe of the basal ganglia results
in increased inhibition of the subthalamic nucleus, which
in turn results in disinhibition of thalamocortical fibres.
Several studies have suggested that cannabinoid receptor
activation may inhibit the release of GABA from projection terminals, 41,64 thus loss of cannabinoid receptors may
result in increased GABA release within these regions,
which may compensate for the initial loss of GABAergic
functioning. That alterations in cannabinoid receptor levels
may significantly alter other neurochemistry was clearly
demonstrated recently in the production of a mouse lacking
cannabinoid receptors; 62 these animals demonstrated
increases in substance P, dynorphin and enkephalin in the
caudate nucleus and putamen.
Alternatively, while D1 and cannabinoid receptors are
clearly co-localized on striatonigral and striatopallidal projection terminals, it is possible that they display an uneven distribution on these terminals. This study would therefore imply
that medium spiny neurons with a higher ratio of cannabinoid
to D1 receptors are preferentially degenerating in early
Huntington’s disease. Cannabinoid compounds such as the
non-psychotropic HU-211 have been demonstrated to be
neuroprotective; 13,44,68 however these compounds do not
activate the CB1 receptor. A recent study demonstrated that
tetrahydrocannabinol exposure can lead to cell death via the
CB1 receptor; 8 high levels of cannabinoid receptors may
therefore render the cells more sensitive if the disease process
has resulted in increased levels of endogenous cannabinoid
agonist as has been recently reported for schizophrenia. 36
Furthermore, any increase in endogenous agonist level
could result in a down-regulation of CB1 receptors. A
down-regulation in cannabinoid receptors in response to
chronic exposure to cannabinoids has been demonstrated
previously. 45 We are currently investigating the levels of
Receptor alterations in Huntington’s disease
517
Fig. 11. Schematic summary diagrams demonstrating the relationship of the alterations in receptor binding to the pattern of degeneration of neurons in the basal
ganglia in Huntington’s disease. (A) demonstrates the neuronal localization of receptors in the normal human brain based on previous studies in animal and
human brains. The findings presented in this study suggest that there are at least three sub-populations of GABAergic medium-sized spiny striatal efferent
neurons: GABA/ENK neurons projecting to the GPe; and two populations of GABA/SP neurons projecting to either the GPi or the SNr. (B) summarizes the
interpretation of the findings in grade 0 Huntington’s disease cases; the early degeneration of GABA/ENK neurons projecting to the GPe is suggested by the
loss of receptor binding both within the caudate nucleus and putamen and in the GPe. Furthermore, the selective loss of cannabinoid receptor binding at grade 0
in both the GPi and the SNr in the presence of normal D1 receptors suggests that these terminals are still intact. (C) demonstrates the findings in grade 1
Huntington’s disease: the degeneration of GABA/SP neurons projecting to the SNr is indicated by the loss of both cannabinoid and D1 receptor binding in
the caudate nucleus and putamen and the SNr; also, of note is the further loss of cannabinoid receptors within the GPi, without the loss of D1 receptors in
this region, suggesting that the terminals are still intact in the GPi. (D) demonstrates that the results of the binding studies suggest that by grade 3
Huntington’s disease all pathways show advanced degeneration. The receptors lost in the various grades of Huntington’s disease are outlined in black in
B, C and D.
the endogenous agonists anandamide and 2-arachidonyl
glycerol in these brains. Interestingly, a recent study
demonstrated an increase in anandamide levels in the globus
pallidus of reserpine-treated rats, which is a model of
Parkinson’s disease. 31
Whether these various neurochemical changes are occurring in response to the disease process or are contributing to it
is unclear. It is not yet possible to further elucidate the
mechanisms involved here until the function of the Huntington’s
disease gene, 65 and the endogenous cannabinoid ligands are
better understood. While the mechanism and significance of
the cannabinoid receptor loss is speculative at present, this
study suggests that selective vulnerability does exist among
medium spiny neurons to the degenerative processes in
Huntington’s disease. Furthermore, this study emphasizes
that the degeneration of terminals and receptors are not
necessarily parallel processes. The findings here demonstrate
the novel finding that cannabinoid receptor binding declines
518
M. Glass et al.
dramatically in early grade Huntington’s disease, prior to
the apparent degeneration of the terminals as indicated by
co-localized receptors. These changes may indicate that
cannabinoids have a central role in the progression of neurodegeneration in Huntington’s disease.
Acknowledgements—This study was supported by grants from the
Health Research Council of New Zealand, the New Zealand
Neurological Foundation and the New Zealand Lottery Board.
Michelle Glass was supported by the J. B. Miller Postgraduate
Scholarship from the New Zealand Neurological Foundation
Inc.
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(Accepted 7 January 2000)